ES2226331T3 - MEDICINAL CONTAINING OLIGOSACARIDS FOR THE TREATMENT OF APOPTOSIS DYSFUNCTIONS. - Google Patents

Abstract

Medication, characterized by presenting, as active ingredient, an effective amount of at least one oligosaccharide substance appropriate to modulate apoptosis dysfunctions and which eventually, at least in certain of its unit motifs, at least one substituent of the group that it comprises the sulfate, methyl and acetyl groupings, the substance being chosen in the group comprising - the enzymatically or chemically derived oligosaccharides of the polymers of the group comprising beta 1-3 glucans that eventually carry beta 1-6 branches, and and - the enzymatic or chemical derived oligosaccharides of carrageenans, agars and porphranos.

Description

Medication containing oligosaccharides for Apoptosis dysfunction treatment.

The present invention aims at a medication for the treatment of apoptosis dysfunctions.

It is designated by the term "apoptosis" the programmed cell death or cell suicide.

This word corresponds to a self-elimination of the cells according to a defined program.

It is initially translated by swelling at the level of the plasma membrane, swelling accompanied by a change structural of the membrane, and then by loss of volume of the cell that seems to contract and sink on itself.

The nucleus condenses and the DNA is cloned or fragmented into small pieces (Raff, "Nature", 356, 397, 1992; Bortner and others, "Trends in Cell. Biol." 5, 21, 1995).

In vivo , the apoptosis cell is recognized by the macrophages that will phagocyte it and eliminate it in the absence of any inflammatory process.

Always in vivo , apoptosis is widely used by living organisms to control cell populations, particularly lymphocytes after activation.

On the other hand, apoptosis plays, in the course of the development of organisms, a primary role in the removal of unnecessary embryonic tissues (lizard tail, rudiment of genital organs of one sex or the other), and in the organism formation (removal of interdigital membranes between future fingers and others).

Certain compounds present in organisms living specifically induce an apoptotic phenomenon. So by example, in mammals, the Fas ligand binding to the membrane Fas receiver, also designated by APO-1 or CD95, specifically induces apoptosis; this apoptosis is used by the living organism to control populations of lymphocytes, especially T lymphocytes.

Said receptor and ligand represent a system extremely interesting physiological that is involved in the specific removal of cells that are no longer desirable in the organism.

You can cite in particular the elimination cell in the course of maturation and activation of T lymphocytes. Actually, the Fas system, that is, ligand Fas / Fas receptor, plays a primary role in homeostasis of the immune system.

The Fas receiver is a member of a family of proteins that act as receptors on the surface of cells and which also comprise TNF receptors (necrosis factor tumor) and NGF (nerve growth factor).

The Fas receiver is expressed in numerous cells; will accumulate at the Golgi apparatus level.

The mechanism by which the Fas system induces cell death is not known but requires the activation of known proteases with the designation "similar to ICE" ("interleukine-1 beta-converting enzyme ") in English) or caspases.

It can be seen that the Fas ligand can be secreted by cells to induce their own suicide; but given that this ligand is also on the surface of the activating cells, these will induce the suicide of trap cells by simple contact. Once activated, the receiver Fas interacts with numerous intracellular proteins to transmit the signal that initiates apoptosis.

In vitro , there are other means to induce apoptosis, for example, by inhibiting the activity of certain kinases, and in particular kinase C; In this case, staurosporine can be used.

This product is very effective in inducing death of cells due to apoptosis.

On the other hand, EP 795 560 describes a keratan oligosaccharide sulfate as an agent capable of inducing apoptosis

However, it should be noted that transduction of the signals induced by staurosporine is different from the which makes the Fas receiver intervene.

However, while the means of activating the apoptosis are different, the execution of the death program induced by these two modes of activation is equivalent and it characterized by an activation of the caspases cascade and a mitochondrial dysfunction that releases compounds (for example, cytochrome C) that will promote the programmed destruction of the cell. This phenomenon has energy dependence but does not require synthesis of new proteins. In reality, everything is found arranged in a cell to ensure its own destruction.

In vivo , the regulation of the apoptotic phenomenon is of considerable importance.

Indeed, numerous pathologies are associated with your dysfunction

One can cite, for example, two cases of apoptosis dysfunction in which it is modulated by intermediate of the Pas system: these are diseases autoimmune diseases in which apoptosis is deficient and the destruction of CD4 + T lymphocytes infected by HIV-1 in which apoptosis is too much active

In other cases, such as degenerations neuronal found, for example, in sclerosis in plaques, apoptosis is activated by pathways that are not yet known.

There are other pathologies in which apoptosis it's hard; in this regard, the accumulation of cancer cells whose apoptosis would appear to depend on the FAS system ("Green", Science, vol 278, 1246, 1997).

The requesting company, in view of the checks that have been indicated above, you have had the merit of discovering that, from the moment you have a medication capable of modulating apoptosis dysfunctions both from the point of view of activation as in the case of pathologies from the group of those who understand autoimmune diseases and cancer-like pathologies and from the point of view of their inhibition in the case of group pathologies of which understand AIDS, it has been possible to fight against these diseases.

The company itself has had the merit no less important to discover that certain substances of the type of oligosaccharides and monosaccharides eventually present, at least in some of its unitary motives, at least one substituent of the group comprising sulfate, methyl and acetyl groups, which are proper to modulate apoptosis dysfunctions.

The invention therefore has as its object a medicine that is characterized by behaving, by way of principle active, a minimum effective amount of a substance of appropriate oligosaccharide to modulate dysfunctions of the apoptosis, and eventually present, at least in some of its unit motives, at least one substituent of the group that it comprises the sulfate, methyl and acetyl groups, said said substance within the group that comprises

- enzymatically derived oligosaccharides or chemistry of the polymers of the group comprising? 1-3 glucans that eventually present ramifications? 1-6,

- enzymatically derived oligosaccharides or chemistry of carrageenans, agars and porphranos.

According to an advantageous embodiment, the medicament according to the invention presents, by way of principle active, an effective amount of at least one oligosaccharide appropriate to modulate apoptosis dysfunctions and that answer the formula

one

in which n represents a number integer from 1 to 50, preferably from 5 to 10 and in which the number of ramifications varies from 0 to 3 per unit of repetition.

According to another advantageous embodiment, the medicament according to the invention presents, by way of principle active, a minimum effective amount of a disaccharide of appropriate repetition to modulate apoptosis dysfunctions and corresponding to the formula

2

in which n represents a number integer from 1 to 50, preferably from 1 to 20, being able to present as minimum any of the repeat disaccharides of formula (II) one or several groups sulfate.

According to another advantageous embodiment, the medicament according to the invention presents, by way of principle active, an effective amount of the appropriate product to inhibit as partially minimal apoptosis and that is obtained by hydrolysis to from sodium iota-carrageenan, being constituted this product by a mixture of oligo-iota-carragenanos designated per I_ {g}, whose content in total bears (determined according to Tillmans and Philippi) is 62% and whose distribution profile for size, estimated by electrophoresis on polyacrylamide gel according to Zablakis and Perez, is:

3

These methods are described, as regards to Zablakis E. & Perez J., in "Botanica marina", 33, 273-276 (1990) and, as regards Tillmans J. and Philippi K., in "Biochem. Z", 215, 30-60 (1930).

To prepare the product I g, you can proceed as follows.

The iotacarrageenan is incubated in the presence of the partially purified iotacarragenase enzyme at a temperature of 45-50 ° C, and then the hydrolysis products are ultrafiltered on a 10,000 Da membrane. You get this way the product I_ {g}.

More particularly, the iotacarrageenan polymer is hydrolyzed by a recombinant iotacarrageenan expressed in the Escherichia coli strain.

The enzyme preparation is done by dissolution of the bacterial residue (corresponding to one liter of culture) in 50 ml of 10 mM Tris buffer pH 7.5, 100 mM NaCl, 5 mM CaCl2 so that at the end, 500 U / ml is obtained.

From a practical point of view, they dissolve 100 g of iotacarragenano substrate in 20 l of distilled water in hot (80 ° C) so that a concentration of 5 g / l is obtained and then the pH is adjusted to 7.5 with ammonium carbonate.

To effect hydrolysis, the enzyme is added at 50 U / g of polymer. Continuous ultrafiltration is performed after 30 minutes; in the case of ultrafiltration tangential.

For this tangential filtration, it was possible use a Pellicon brand device that has a cassette 10,000 Da 0.46 m 2 PTGC of the Millipore Society; this apparatus It is set to 2 bar at the entrance and 0.5 bar at the exit.

The filtrate outlet is partially closed to maintain the filtrate flow rate at 1 liter per hour.

The characteristics of the reactive envelope are they choose so that they allow a supply of the enzyme in the substrate until depletion of the 20 liters of solution and the maintenance of a fixed volume of 2 liters.

18 liters of an ultrafiltrate are obtained which is concentrated to 1 liter by rotary evaporation, and then the Concentrate is lyophilized. The lyophilisate obtained in this way contains the product I_ {g}.

The oligocarragenanos of fraction I_ {g} obtained in this way have undergone a fractionation supplementary by low pressure chromatography on column Biogel P6 and then on Sephadex G10 column.

You get fractions this way identical before mentioned.

According to another advantageous embodiment, the medicament according to the invention presents, by way of principle active, an effective amount of the appropriate product to inhibit as partially minimal apoptosis, consisting of fraction DP 7 of the product I_ {g}.

According to another advantageous embodiment, the medicament according to the invention has, by way of active principle, an effective amount of the appropriate product to activate apoptosis dysfunctions, obtained by acidic aqueous extraction from a brown algae called Laminaria digitata , whose The product consists of a mixture of oligo? 1-3 glucans designated L11 and having from 1 to 50, preferably from 20 to 30, saccharide units, the product in question presenting the NMR spectrum shown in Figure 1 .

It should be noted that product L 11 can also be obtained by aqueous extraction from brown algae in general, of which Laminaria digitata is a representative.

The preparation of product L 11 can be perform as follows.

To 300 g of fresh seaweed of the Laminaria digitata type collected in the month of August in fresh or dried form, 1 l of 0.3% sulfuric acid is gradually added.

The operation is performed in a water bath at one approximate temperature of 70ºC for 2 hours and 30 minutes with agitation.

This operation is renewed twice.

The extract obtained is clarified by filtration on a 1.2 µm porosity filter.

The liquid resulting from this filtration is subjected to tangential ultrafiltration on a membrane of porosity of 50,000 Daltons.

Ultrafiltration is performed by maintaining a 1 bar pressure.

In this way an ultrafiltrate is obtained whose pH is brought to 5.5, presenting an approximate volume of 0.8 liters. This ultrafiltrate is subjected to dialysis on a membrane of cellulose ester with porosity of 500 Daltons.

A dialysate is obtained that is concentrated to a 100 ml volume by evaporation at 80 ° C with the help of a device ROTOVAPOR type, and then it is lyophilized.

7 g of a colored powder material are obtained cream that constitutes the product L 11.

Ion chromatography analysis coupled to amperometry and using an ion exchange resin of DIONEX society shows that oligo? 1-3 glucans constituting said material in powder actually have 1 to 50, preferably 20 to 30, saccharide units.

Chromatographic conditions (method called HPLC, that is, "Chormatographie liquide sous haute pression" (High pressure liquid chromatography)) are the following:

4

the curve that was obtained shown in figure 16 and that identifies the product L_ {11}.

Examination of the 13 C NMR spectrum of product L 11, performed from a solution at 80 mg / ml in D 2 0 and depicted in Figure 1, shows a β-D skeleton - (1 → glucan) whose resonances of the different carbons have been identified (they have been gathered in the following table A) by comparison with the values of the known literature (see Williams et al., 1992 "Development of a water-soluble , sulfated (1 → 3) -? -D-glucan biological response modifier derived from Saccharomyces cerevisiae " , Carbohydr. Res. 235: 247: 25).

TABLE I

5

The medicaments according to the invention defined previously they present the classic formulation aids that correspond to their form of administration and dosage chosen

A subject of the invention is also a method of preparing a medicament for the treatment of apoptosis dysfunctions, characterized in that at least one of the active ingredients identified above is made to behave in a galenic composition.

According to an advantageous embodiment, said galenic compound is appropriate for administration via intravenous

The invention also relates to the use, for the purpose of preparing a medicament for treatment of apoptosis dysfunctions of saccharide substances from the group comprising oligosaccharides derived via enzymatic or chemical polymers of the group comprising β 1-3 glucans that eventually behave Branches? 1-6, and oligosaccharides enzymatic or chemical derivatives of sulfated galactans, especially the carragenanos, the agares and the porfiranos.

More particularly, it refers to the use, for the purpose of preparing a medicine for a treatment of apoptosis dysfunctions of the oligosaccharides of formula (I) and those of formula (II).

More particularly still, it refers to the use of the products indicated by I_ {g} and L_ {11} and of the DP 2 and DP 7 fractions of I g for the purpose of preparing medicines for the treatment of dysfunctions of the apoptosis

The invention will be better understood with the help of the following description complement and of the examples that do not they are limiting but correspond to embodiments advantageous

In the experiences described in then, we have worked on cell cultures in which an apoptotic process has been caused having resorted to the system Fas or staurosporine, and then the modulation effects that can be obtained with the products that they constitute the active substance of medicines according to the invention.

Within the framework of these experiments, it has been determined, on the one hand, the appropriate amounts of principle active to obtain the desired effect of apoptosis modulation and, on the other hand, the moment or moments in which it is convenient administer, to obtain the desired modulation effect, the active substance or the medicine that presents it.

Example 1

Work has been done on a fibroblast culture of genetically modified mice to express constitutively the human Fas receptor; the controlled active substance was the product designated by Ig.

In previous experience it has been shown that murine fibroblasts are destroyed by apoptosis when they put in the presence of the Fas ligand or an agonist antibody that recognizes the Fas receiver and designated FasAb below.

In another previous experience, it has been determined that the product I g did not alter cell growth, which was not toxic with respect to murine fibroblasts in concentrations used and that could therefore be added to a means of cell culture without creating problems.

The medium used for the cultivation of Murine fibroblasts is marketed by the Life Society Technologies with the designation "Modified Eagle Medium of Dulbecco ", this medium having been described in" Virology "8, 396 (1959) by Dulbecco and others.

5% by volume of serum has been added to this medium cow fetal

This medium has been inoculated with murine fibroblasts in the presence of a sufficient amount of antibiotics to eliminate the possibility of contamination; the Concentration of the culture medium in fibroblasts has been 10 5 cells per ml of the medium.

The cultivation has been carried out inside a incubator in which the temperature has been maintained at 37 ° C; containing the atmosphere that filled the incubator 5% of CO 2.

After incubation for 24 hours, it has added the Fas ligand, or the FasAb directly in the means, medium.

The amount of FasAb that has been added has been 50 µg per ml of culture medium.

Under these conditions, approximately 70% of culture cells are destroyed by apoptosis after approximately 24 hours of incubation.

This destruction is demonstrated by the coloring to the violet crystal of the surviving cells.

A certain number has been followed of essays aimed at highlighting the action of the principle active.

In these trials, it has been varied, by a part, the concentration at which the active substance is used in the culture medium and, on the other hand, at the moment in which the active substance is added to this medium so that they are determined the optimal concentrations in active principle, as well as the moment or the most opportune moments of introduction of the active substance regarding the addition of FasAb.

The concentrations of active substance of 0.001 to 2 mg per ml.

The effect obtained has been studied successively, initially adding the active substance before FasAb, to continuation at the same time and finally after FasAb.

In a first essay, the principle has been added active 24 hours before FasAb.

Within the framework of this essay, the effect obtained, using successively 0, then 5, a continued 10, then 50, then 100 and finally 500 ng of FasAb per ml of culture and, in each case, concentrations of active substance successively equal to 0.25, then 0.5 and finally 1 mg per ml of culture medium, it should be understood that the effect obtained in the absence of active substance, that is, for a concentration of 0%.

After 24 hours of incubation, proceed to survival analysis

The result of this analysis is materialized by the histogram of figure 2.

On the abscissa axis of this histogram, the concentration of the medium in FasAb is expressed in ng / ml and, in an axis of ordinates, the proportion of survival expressed in %.

For each of the concentrations in FasAb, has materialized the survival rate for each of the four concentrations of active substance, ie 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml, for four rectangles parallel to the axis of ordinates, materializing the displacement type each time with a segment that exceeds the corresponding rectangle parallel to the axis of the ordinates.

The rectangle corresponding to 0 mg / ml of active ingredient is, in each case, the one located more towards the left, which corresponds to 0.25 mg / ml of active substance is located on the right, which corresponds to 0.5 mg / ml is located at the right of the previous one and so on.

The four rectangles are identified each time by grated, specific points or marks.

The examination of the results gathered in this way in the histogram of figure 2 shows that in the absence of active principle, the survival rate decreases with the increased FasAb concentration and that this ratio of survival is significantly improved by adding principle active.

In a second trial, the means of Cultivation of FasAb and the active substance at the same time.

In this second trial, the effect has been observed obtained using successively the same concentrations in FasAb and in principle active than in the first trial.

After 24 hours of incubation, it has proceeded to survival analysis.

The recorded results have been collected in the histogram of figure 3 which is constituted according to them principles that have been exposed with respect to figure 2.

The examination of these results demonstrates that the survival rate evolves analogously to what is observed in the first trial

In the third series of trials, the active substance after FasAb, that is, successively:

at the beginning, 1 hour after FasAb,

then 3 hours after FasAb, and

finally, 6 hours after FasAb,

varying in all cases FasAb concentration from 0 to 500 ng per ml of culture.

In the case of the addition of active substance made 1 hour after FasAb,

-

It has been gathered on the histogram of figure 4 the indicated results for concentrations in I g of 0 mg / ml of 0.005 mg / ml, of 0.01 mg / ml and finally 0.05 mg / ml,

-

they have gathered on the histogram of figure 5 the indicated results for concentrations in I g of 0 mg / ml, of 0.1 mg / ml, of 0.25 mg / ml and finally 0.5 mg / ml, and

-

they have gathered on the histogram of figure 6 the indicated results for concentrations in I g of 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml and finally 1 mg / ml.

The histograms of figures 4 to 6 are constituted according to the same principles as those exposed to purpose of figure 2.

In the case of the addition of active substance made 3 hours after the addition of FasAb, has met on the histogram of figure 7 the results indicated for I g concentrations of 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml and of 1 mg / ml.

In the case of the addition of active substance made 6 hours after the addition of FasAb, has met on the histogram of figure 8 the results indicated for I g concentrations of 0 mg / ml, 0.25 mg / ml, 0.5 mg / ml and of 1 mg / ml.

The histograms of Figures 7 and 8 are constituted according to the same principles as those exposed to purpose of the corresponding figure 2.

The overall review of the results gathered on the histograms of figures 4 to 8 shows that, in the case of the addition of the active substance after the addition of FasAb, the survival rate always increases; this effect has always a tendency to decrease when the elapsed time increases between the successive additions of FasAb and active ingredient; by On the other hand, it is sensitive to the concentration in active principle when it is less than 0.25 mg / ml; no improvements are obtained sensitive for concentrations greater than 0.25 mg / ml.

In the experiment described, the Apoptosis was induced by the FasAb system.

Another experience has been made inducing apoptosis by the kinase inhibitor constituted by the Staurosporine

It has been recalled that staurosporine induces apoptotic death in the case of most dose cells ranging from 0.5 to 5 µM.

In this experiment, the addition of staurosporine and of I_ {g} was simultaneous.

Two doses of I g have been used, namely, 0.2 mg / ml and 0.5 mg / ml.

Staurosporine has been used at a rate of 0.5 µM and then 1 µM and finally 1.5 µM.

The survival rate of the cells treated has been determined in each case 18 hours after incubation.

The recorded results appear in the histogram depicted in figure 9 showing the proportion of Survival expressed in% depending on the concentration of Staurosporin and for the doses of I_ {g} identified previously.

A witness experience has been carried out for a 0 µM staurosporine concentration.

The examination of the results that appear in the histogram demonstrates that the use of I_ {g} attenuates the Staurosporine-induced apoptosis.

It results from the two experiences that have been described that the medicament according to the invention allows obtaining a significant attenuation of apoptosis when it is induced by Different agents

Example 2

In this example, the cell culture studied was a culture of immortalized human cells, consisting of T lymphocytes (Jurkat type).

The culture medium used is the marketed by the company Life Technologies with the designation "RPMI 1640 Medium"; This medium is described by Moore and others, in the publication "A.M.A." 199,519 (1967).

10% by volume of serum is added to this medium fetal cow and antibiotics to eliminate the chances of pollution.

This medium is inoculated with a quantity of 10 6 T lymphocytes per ml of medium.

The incubation temperature is 37 ° C and the atmosphere that fills the incubator contains 5% CO2.

After a 24 hour incubation, they are added Simultaneously FasAb and the active substance.

The amount of FasAb (this is manufactured by the Upstate Biotechnology Society and marketed under the number of catalog 05-201 by the Euromedex Company) added It is 50 ng / ml of culture.

The active substance is constituted successively by the product I_ {g} and the product L_ {11}.

The amounts used are, in each case, of 0.5 mg / ml

Survival analysis is performed 18 hours After the start of the experiment.

This survival analysis consists of doing pass a volume of culture containing 10 4 cells in a apparatus of the type that works by flow cytometry, in this case is marketed by the Beckton Dickinson Society with the designation "FAC Scan cytometer".

This device uses a probe that detects the presence of phosphatidyl serine on the surface of the cells; the Presence of this product shows that the cells in question are apoptotic

The results of this analysis appear in the Figures 10 to 13 in each of which three have been represented polygons, respectively (A), (B) and (C) whose contours have been defined to be representative of cell populations different; polygon (A) encloses a set of living cells, the polygon (B) a set of apoptotic cells and polygon (C) a set of dead cells.

In the following table II the percentages of living cells, apoptotic cells and the percentage of dead cells in the case of each of figures 10 to 13 which will be commented later.

TABLE II

6

Figure 10 shows the survival analysis made on a sample of a culture medium in which it has not been FasAb added nor active ingredient; it is a witness; in this case, it is appreciated that there are substantially only living cells found in polygon (A) (see line 1 of the table II).

Figure 11 shows the survival analysis carried out on a sample of a culture medium, in which it has not been added more than FasAb; in this case, it is appreciated that the polygon (B) contains 21% apoptotic cells (see line 2 of the table II).

Figure 12 shows the survival analysis made on a sample of a culture medium that has received the simultaneous addition of FasAb and the active substance I g (0.5 mg / ml); in this case, it can be seen that polygon (B) does not contain practically apoptotic cells (2%), polygon (A) contains many living cells and polygon (C) a certain concentration (6%) of dead cells (see line 3 of table II).

Figure 13 shows the survival analysis made on a sample of a culture medium that has received the Simultaneous addition of FasAb and the active substance L 11 (0.5 mg / ml); in this case, it is appreciated that the polygon (B) contains a significant amount (13%) of apoptotic cells and polygon (C) a non-negligible amount (9%) of dead cells (see row 4 of table II).

The conclusions that can be drawn of the examination of figures 10 to 13 and of the analysis of the percentage of cells present in the different polygons do, as consequently, that the active substance I_ {g}, within the framework of this experience, inhibit FasAb apoptosis (from 21% to 2% of apoptotic cells). However, there is a slight increase in number of dead cells (this number goes from 1% to 6%). Whether consider the number of living cells, a protection of 13% (from 77% to 90%).

As regards the active substance, L_ {11}, an increase in the number of dead cells is observed (from 1% to 9%) with respect to the effect induced by FasAb only. He principle L_ {11} although it does not induce an important effect on the number of living cells, would therefore act as an enhancer of cell death

In order to optimize the action of L 11, the following experiments have been performed.

It is administered, using the same culture as previously:

\ ding {226}

in the first case, 50 ng / ml from FasAb alone (from Euromedex identified more above)

\ ding {226}

in a second experiment, the same amount of the same FasAb simultaneously with 0.5 mg / ml of product L_ {11} and

\ ding {226}

in a third experiment 0.5 mg / ml of product L 11 and subsequently, 24 hours later, 50 ng / ml of the same FasAb.

The results obtained after 18 hours of incubation are as follows compared to what was observed on a control culture that has not received the FasAb addition or from L_ {11}, the magnitude taken into consideration being the number of living cells:

\ blacktriangleright

in the case of the FasAb additive only: the number of living cells decreases in 3.6%,

\ blacktriangleright

in the case of the Simultaneous addition of FasAb and L 11, the number of living cells decreases by 6.4% and

\ blacktriangleright

in the case of deferred addition of FasAb and L 11, the number of living cells decreases by 13.7%.

It follows from the above that L_ {11} is much most active when administered before FasAb.

It is indicated that similar results have been obtained replacing the FasAb identified above with a FasAb of another origin, that is, the one manufactured by the Alexis Society Corporation (San Diego, U.S.A.) and marketed by the Company Take S.A. (Paris).

From the examination of the results of the experiments above, it turns out that a medicine based on the product L 11 allows to enhance apoptosis; therefore you can foresee your use in the treatment of autoimmune diseases and Of cancer.

Other experiments have allowed verifying the effects produced by the administration of the product I_ {g}, by one part, in lymphocyte cultures in which a Fas apoptosis of low intensity and, moreover, in the case of lymphocyte cultures in which Fas apoptosis has been induced of strong intensity.

Fas apoptosis of low intensity may be induced using a FasAb of Euromedex origin; an apoptosis This type can be of the order of 3 to 10%.

Fas apoptosis of strong intensity can be induced using a FasAb of origin Alexis Corporation; a Apoptosis of this type can be of the order of 20 to 50%.

Experiences made using a dose 0.2 mg / ml of the product I g have been shown in Figure 14 which shows the recorded effects, that is, the stimulation or the inhibition of apoptosis (expressed in%) depending on the intensity of apoptosis (in%) induced only by FasAb (concentration of 50 to 200 ng / ml).

Figure 14 shows that

\ blacktriangleright

in the case of Low intensity apoptosis, of the order of 3 to 10%, materialized on the axis of abscissa by points a, b, c, d, e, f, g, h, i, the administration of I_ {g} causes an enhancement of 20 to 40% of the apoptosis with respect to FasAb apoptosis only and

\ blacktriangleright

in the case of apoptosis of strong intensity, of the order of 20 to 50%, materialized on the axis of abscissa points l, m, p, q, the administration of the same amount of I_ {g} causes a 5 to 20% inhibition of apoptosis with respect to apoptosis Produced by FasAb only.

Knowing that it is possible to determine in a subject The induced apoptosis at the lymphocyte level is determined. therefore it is possible to modulate this according to the proven intensity of said apoptosis in the sense of potentiation or in the sense of inhibition by administering to this subject a medicine based on I_ {g}.

As a consequence, this medicine may allow the correction of apoptosis in the sense of potentiation when the subject is affected by a disease of cancer or autoimmune disease type corresponding to a reduced apoptosis and in the sense of an inhibition when the subject suffers a syndrome type condition immuno-deficient corresponding to an apoptosis strong.

In this situation, it is required that the lymphocytes Jurkat type T present in the cultures subjected to the experiments above, whether cells whose tumor suppressor gene p53 or protein p53 (Bing An et al., 1998, "Cell Death and Differentiation" 5, 1062-1075) has mutated and, because of this, is inactive.

The effects observed with the products used according to the invention, especially with I g and L 11, they are probably independent of p53; in other words, the products I_ {g} and L_ {11} are able to enhance the stimulation or inhibition of apoptosis by its unique presence and regardless of the presence or absence of the p53 gene active.

However, in most cancers humans (see Hollstein et al., 1994, "Nucl. Acid Res.", 22, 3551), the p53 protein has mutated, and therefore is inactive; be it follows that the use of a medicine based on one of the products according to the invention, and in particular the products I_ {g} and L_ {11}, allows to solve the dysfunctions of the p53 protein and treat cancer-like diseases and autoimmune diseases.

The invention allows, as a consequence, to treat the diseases in question in a fundamentally different way and simpler than gene therapy that is based on the principle that especially refers to the reintroduction of a normal p53 gene in the genome of the mutated cells of a sick subject.

It is known that most of the cancer cells have lost their sensitivity to FasAb apoptosis and that most of the anticancer agents currently used act by activating the p53 gene which, in turn, acts positively on the FasAb system. However, these systems stop working if there is a p53 mutation (Müller et al., 1998, J. Exp., Med. 188, 2033-2043). According to recent observations, the FasAb system is essential for the destruction of cancer cells, either by the immune system or by the action of anticancer drugs; it follows that the fact that the products used according to the invention and especially I_ {g} and L_ {11} modulate the apoptosis of said cancer cells independently of p53 is of considerable importance and allows for the use of a new drug generation especially
anti-cancer

Example 3

The product I_ {g} is composed, as it has been indicated above, of a mixture of oligo-iota-carragenanos.

The effect induced by different constituents of this mixture, in particular of DP 2, DP 3, DP 4, DP 5 and DP 7 fractions.

Likewise, the polymer that constitutes the raw material of I_ {g}, as well as, the product called KIK (hexasaccharide type kapa-iota-kappa).

For these experiments, we have proceeded as indicated in example 1, entering simultaneously, by one part, 0.2 mg / ml of each of said I g fractions and, on the other, 100 ng / ml of FasAb of Euromedex origin.

After 24 hours of incubation, they have been done the checks that are summarized below.

The DP 2, DP 3, DP 4 and DP 5 fractions are inactive and do not stimulate dose-induced apoptosis reduced (100 ng / ml) FasAb from Euromedex origin inducing by itself 6% cell death after 18 hours of incubation.

On the contrary, an effect is obtained very strong stimulator for the DP 7 fraction (50%) and for the product I_ {g} (70%), which shows that one of the active components of the mixture I g may be constituted by fraction DP 7; by On the other hand, the KIK product has no activity.

Finally, it has been discovered that the polymer that constitutes the raw material for the preparation of I_ {g} before fractionation or clivated by the enzyme iotase is, for its part, active and stimulates Fas apoptosis of reduced intensity. For him On the contrary, the above iotasa, which in turn is used in 50 units, has no activity when incubated with cells.

It follows that the active substance I_ {g}, which has no intrinsic apoptotic activity, induces a very strong stimulation in the case of a FasAb apoptosis of reduced intensity

Example 4

A complementary experiment has been carried out which confirms the results translated by figure 14.

In the case of induced apoptotic phenomena by Fas, the first of the caspases of the caspasas cascade activated is caspase 8.

Measurement of the activity of this caspase 8, which is a protoelitic enzyme, after lysis of Jurkat cells treated or not with FasAb only and with FasAb at the same time than with I_ {g}.

For this, the measurement of

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the activity of the caspase 8 in a first fraction of a Jurkat cell culture in absence of FasAb and I_ {g}; the percentage of living cells in the crop is 93%;

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the activity of the caspase 8 in a second fraction of the same crop, 18 hours after incubation with 500 ng / ml FasAb of origin Euromedex (which, as indicated above, induces a reduced apoptosis, of the order of 13%); being the percentage of live cells in the 80% culture;

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the activity of the caspase 8 in a third fraction of the same crop, 18 hours after incubation with 500 ng / ml FasAb of origin Euromedex and 0.2 mg / ml of I g; being the percentage of cells live in the 50% culture;

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the activity of the caspase 8 in a fourth fraction of the same crop, 18 hours later of incubation with 25 ng / ml of FasAb from Alexis (which, as indicated above, induces an intense apoptosis, of the order of 40%); the percentage of live cells in the culture is of 53%;

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the activity of the caspase 8 in a fifth fraction of the same crop, 18 hours later of incubation with 25 ng / ml of FasAb from Alexis and 0.2 mg / ml of I g; being the percentage of living cells in the 59% culture;

The measurement of caspase activity has been made each time with the help of a team or kit marketed by the company Ozyme under the name "Apo Alert Flice Fluor "N0 K2028-2.

The results of these measurements have been collected in the histogram of figure 15.

The examination of this histogram shows that

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caspasa 8 activity is weakly stimulated (close factor of 1.37) by FasAb Euromedex only, inducing the simultaneous administration of I_ {g} a strong stimulation (close factor of 3.93),

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caspasa 8 activity is strongly stimulated (close factor of 4.96) by FasAb Alexis only, decreasing this strong stimulation (factor close to 4.05) when I_ {g} is entered simultaneously with FasAb Alexis

There is, as a consequence, a net correlation between apoptosis and caspase activity 8.

The product I_ {g} therefore represents a product capable of modulating the activity of caspase 8.

The fact that this enzyme is shown to be found essentially in the biochemical pathway of apoptosis induced by Fas (Juo et al., Curr. Biol. 8, 1001-1008, 1998), allows to anticipate new medications based on oligosaccharides that allow to capture type enzymes Caspase

These products therefore represent a alternative to a therapy based on peptides that carry the sequence recognized by caspase 8 ("suicide substrate" of caspase 8).

Claims (10)

1. Medication, characterized by presenting, as active ingredient, an effective amount of at least one oligosaccharide substance suitable for modulating apoptosis dysfunctions and which eventually, at least in certain of its unit motives, at least one substituent of the group comprising sulfate, methyl and acetyl groups, the substance being chosen in the group comprising - enzymatically derived oligosaccharides or chemistry of the polymers of the group comprising? 1-3 glucans that eventually behave ramifications? 1-6, and - enzymatically derived oligosaccharides or chemistry of carrageenans, agars and give us 2. Medication, characterized by carrying, as active ingredient, a minimum effective amount of an oligosaccharide appropriate to modulate apoptosis dysfunctions and responding to the formula 7 in which n represents a number integer from 1 to 50, preferably from 5 to 10 and in which the number of ramifications varies from 0 to 3 per unit of repetition. 3.Medicament, characterized by presenting, as active ingredient, an effective amount of at least one repetitive disaccharide appropriate to modulate apoptosis dysfunctions and responding to the formula 8 in which n represents a number integer from 1 to 50, preferably from 1 to 20, being able to at least some of the repeat disaccharides of formula (II) present one or more groups sulfate. 4. Medication, characterized in that, by way of active principle, an effective amount of the appropriate product to partially inhibit apoptosis and which is obtained by hydrolysis from sodium iotacarrageen, this product being constituted by a mixture of oligo-iota - carrageenans indicated by I_ {g}, whose content in total bears (determined according to Tillmans and Philippi) is 62% and whose distribution profile by measure, estimated by electrophoresis on polyacrylamide gel according to Zablakis and Pérez, is: 9 5. Medication, characterized by carrying, as active ingredient, an effective amount of the appropriate product to activate apoptosis dysfunctions, obtained by acidic aqueous extraction, from brown algae and more particularly from a brown algae called Laminaria digitata , whose product is constituted by a mixture of oligo? 1-3 glucans designated by L11 and having from 1 to 50, preferably from 20 to 30 units of saccharides, the product in question presenting the NMR spectrum shown in Figure 1. 6. A medicament, characterized in that it comprises, as an active ingredient, an effective amount of the appropriate product for activating apoptosis dysfunctions and consisting of the DP7 fraction of the product I g according to claim 4. 7. Procedure for the preparation of a medicament for the treatment of apoptosis dysfunctions, characterized by the fact that a galenic compound is caused to behave at least one of the active ingredients of the medications according to at least one of the claims 1 to 6. 8. Use, for the preparation of a medication for the treatment of apoptosis dysfunctions, at least one of the oligosaccharide substances that have eventually, at least on some of his unitary motives, at least one substituent of the group comprising the groups sulfate, methyl and acetyl, choosing the appropriate substances to modulate apoptosis dysfunctions in the group that understands - enzymatically derived oligosaccharides or chemistry of the polymers of the group comprising? 1-3 glucans that eventually behave ramifications? 1-6, and - enzymatically derived oligosaccharides or chemistry of carrageenans, agars and please tell us, 9. Use, for the preparation of a medication for the treatment of apoptosis dysfunctions, of the oligosaccharides of formula (I) and those of formula (II). 10. Use of the products indicated by I_ {g} and L_ {11} and of the product constituting fraction DP 7 of the product I g, according to claim 4, for the preparation of medications for the treatment of dysfunctions of the apoptosis